Chronic myeloid leukemia (CML) is caused by the reciprocal translocation t(9;22)(q34;q11), resulting in the BCR-ABL1 fusion gene. BCR-ABL1 tyrosine kinase inhibitors (TKIs) improve overall survival in patients with chronic phase CML (CML-CP). Approximately half of the patients who achieve a durable deep molecular response can achieve sustained treatment-free remission (TFR) after TKI discontinuation; thus TFR is now a therapeutic goal for most patients with CML-CP. Sensitive BCL-ABL1 transcript detection methods reveal that evidence of residual CML cells remains in patients who achieve sustained TFR, indicating that the host immune system protects against CML relapse. The human immune system is composed of innate and adaptive arms. Natural killer cells are major components of the innate immune system, while T cells are major components of the adaptive immune system. Myeloid-derived suppressor cells and regulatory T cells, both suppressors of the immune response, have important roles in the regulation of CML. Here, we review the current understanding of the immune response in CML, especially in TFR.
Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder caused by the oncogenic t(9;22)(q34;q11) chromosomal translocation, known as the Philadelphia chromosome. BCR-ABL1 tyrosine kinase inhibitors (TKIs) improve the overall survival of patients with chronic phase CML (CML-CP) . The measure of treatment success is the reduction of detectable BCR-ABL1 mRNA levels in the blood to a point 4.0 or 4.5 logs below that found before treatment commenced; this is known as a deep molecular response (DMR). Approximately half of CML-CP patients who achieve a durable DMR can maintain molecular remission after TKI discontinuation, and now treatment-free remission (TFR) is one of the therapeutic goals for patients with CML-CP . However, why some patients are at higher risk of molecular relapse which is defined as loss or major molecular response (MMR) or DMR has not been fully elucidated. Evidence of residual CML cells can still be found in patients who achieve TFR using sensitive BCL-ABL1 DNA-based detection method , and, therefore, the host immune system could be apparently acting to prevent CML progression or relapse after TKI discontinuation. Here, we review the immunological analyses in studies examining the discontinuation of TKI treatment for CML-CP.
Summary of recent TKI discontinuation studies
The pioneer TKI discontinuation study was the STop IMatinib (STIM) study , which showed that 41% of patients who received imatinib with a durable DMR for at least 2 years maintained TFR for the next 12 months. Subsequently, the TWISTER study also showed that 42.7% of patients maintained TFR after imatinib discontinuation . Both of these studies indicated that imatinib could be discontinued safely. Next, several studies on the discontinuation of second-generation TKIs (dasatinib or nilotinib) were reported. The DAsatinib DIscontinuation trial (DADI) trial enrolled CML-CP patients receiving second-line dasatinib  and the Stop Tasigna Trial (STAT2) enrolled patients receiving second-line nilotinib . TFR was achieved in 49% (DADI) and 67.9% (STAT2) of patients at 12 months. These results suggested that discontinuation of second-line second-generation TKIs was feasible, and first-line second-generation TKI discontinuation trials were also instigated. The first-line DADI trial enrolled CML-CP patients who achieved a DMR with first-line dasatinib treatment for at least 24 months , and the ENESTfreedom trial enrolled patients who achieved a DMR with first-line nilotinib treatment for at least 24 months . After 1 year as consolidation treatment, the dasatinib or nilotinib was discontinued and 55.2% (first-line DADI) and 51.6% (ENESTfreedom) of patients were in TFR. The largest European Stop Tyrosine Kinase Inhibitor (EURO-SKI) trial enrolled 758 CMP-CP patients with a DMR maintained for 1 year during treatment with any TKI, and the TFR rate was 56% at 12 months after treatment ceased . These studies suggested that discontinuation was a safe procedure with imatinib, dasatinib, and nilotinib (Fig. 1). Detailed results of TKI discontinuation trials are summarized in Table 1.
Innate and adaptive immunity in humans
The human immune system consists of innate and adaptive arms, characterized by their functional diversities. Natural killer (NK) cells, a subset of large lymphocytes, play important roles in innate immunity against viruses or tumors by secreting cytotoxic granules  and eliciting antibody-dependent cellular cytotoxicity by releasing cytotoxic cytokines, which activate macrophages to kill target cells. Meanwhile, the proliferating, antigen-specific T lymphocytes .
On primary exposure to an antigen from a pathogen, naïve T cells receive signals via the T-cell receptor (TCR), stimulating their clonal expansion and leading to the proliferation of effector T cells. Through this process, T cells gain appropriate effector function such as lytic activity against virus-infected or tumor target cells . The majority of effector T cells die via apoptosis on completing their action (target cell death). The small minority that survives is defined as memory T cells, which contribute to adaptive immunity.
T-cell immunity in CML
Dasatinib induces large granular lymphocyte expansion associated with favorable outcomes in patients with CML-CP, suggesting that the cytotoxic T cells (CD8+CD57+) may be performing specific anti-CML immune responses. CML-CP patients with increased numbers of peripheral blood CD8+ T cells reportedly have better rates of TFR. The first-line DADI trial showed that a lower proportion of CD4+ T cells at the time of dasatinib discontinuation was associated with a higher rate of TFR , and the DADI trial (be careful, first-line DADI and DADI trial were different trials) also showed that patients with a lower CD4/CD8 ratio tended to achieve a higher rate of TFR . Meanwhile, the D-STOP study showed that a lower proportion of CD4+ T cells was associated with molecular relapse after TKI discontinuation .
Human leukocyte antigen (HLA)-restricted cytotoxic T cells (CTLs) induced by leukemia-associated antigens (e.g., BCR-ABL1, PR1, BMI-1, PRAME, WT1, or CXorf48) are detected in patients with CML, and patients with CXorf48-specific CTLs are more likely to achieve TFR following imatinib discontinuation than those without . We previously showed that certain HLA alleles, which induce antigen-specific CTLs, are associated with the successful achievement of TFR in patients with CML-CP . Increased numbers of the memory CD8+ T-cell (CD8+CD27+CD45RA−) fraction in patients with CML-CP is associated with successfully maintaining TFR , while patients with increased PD-1-expression on CD8+ CTLs (a signature of immune cell exhaustion) are at increased risk of relapse . These results suggested that a specific T-cell population acted in cancer immunosurveillance, contributing to the regulation of leukemic cells in CML, and their exhaustion contributed to leukemia relapse. While the specific constitution of the T-cell population targeting CML cells is likely to play a pivotal role in eliciting a durable TFR in CML, the magnitude of the T-cell proportion required for its maintenance is controversial.
Regulatory T cells in CML
Various immunological self-tolerance systems exist to prevent autoimmune reactions. Regulatory T cells (Tregs) have a pivotal role in preventing excessive autoimmune responses . The differentiation and function of Tregs is regulated by the transcription factor, forkhead box protein 3 (Foxp3). The number of Tregs is significantly increased in patients with CML-CP at diagnosis, indicating that aberrant immune conditions can exist in patients with CML . Myeloid-derived suppressor cells (MDSCs), a heterogeneous population with immunosuppressive activity, mediate the recruitment and expansion of Treg . Imatinib and dasatinib can decrease the number of Tregs and MDSCs in patients with CML, and successful maintenance of TFR is associated with reduced numbers of these populations at the time of TKI discontinuation .
NK-cell immunity in CML
NK cells are components of the innate immune response against pathogens or tumors. Patients with newly diagnosed CML generally suffer from NK-cell dysfunction, whereas treatment with TKIs can restore NK-cell function and this corresponds with molecular remission . Patients with early recovery of the NK-cell population after allogeneic stem cell transplantation have favorable survival outcomes, thus NK cells have a pivotal role in CML regulating leukemic cells.
Surface expression of CD56 divides human NK cells into functionally immature CD56bright and mature CD56dim subsets. CD56bright NK cells can enhance their proliferation and cytokine production, and CD56dim NK cells have more potent cytotoxic functions. In the IMMUNOSTIM and EURO-SKI studies [9, 22, 23], higher numbers of mature NK cells before discontinuation of imatinib were shown to play an important role in the successful maintenance of TFR. By contrast, the D-STOP study showed that patients with a lower number of NK cells at dasatinib discontinuation had a highly durable TFR and the authors propose that small changes in the number of NK cells during the consolidation phase treatment with dasatinib may contribute to TFR . However, the STAT2 and first-line DADI studies reported that increased numbers of NK cells did not affect the successful achievement of TFR [5, 6]. The clinical impact of NK cells in patients who have discontinued TKIs remains controversial.
NK cells are regulated by the balance between activating and inhibitory signals via interactions of their surface receptor molecules with their ligands. Killer immunoglobulin-like receptors (KIR), common NK-cell surface receptors, have abundant structural and functional polymorphisms, which alter NK-cell functions. The KIR2DL5B genotype  or the strong avidity between KIR3DL1 and the HLA-Bw allotype of its HLA class I ligand correlate with a lower probability of DMR  in patients receiving TKI treatment, and thereby also with a lower chance of achieving TFR. Patients who have haplotype A KIR genotype or KIR3DL1 but not the most avid ligand (HLA-Bw4) maintain higher rates of TFR than patients with haplotype B KIR genotype or KIR3DL1 in the context of an HLA-Bw4 .
Immunomodulatory agents in CML for TFR
Only up to 80% of patients with newly diagnosed CML-CP achieve a MR4.5 by TKI monotherapy , and TKI treatment is unable to eliminate quiescent leukemic stem cells because their survival is not dependent on BCR-ABL1 tyrosine kinase activity . Therefore, the combination of TKIs with other types of drug is required to achieve a MR4.5 in more patients (more patients enable to attempt TKI discontinuation) and discontinuation TKI safely. Interferon α (IFNα) stimulates CTLs for the elimination of leukemic cells; it works synergistically with imatinib resulting in higher rates of molecular remission . Furthermore, treatment with IFNα enables discontinuation of imatinib in patients who received prior combination of IFNα and imatinib, and the TWISTER trial showed that patients who receive IFNα treatment achieve a higher rate of TFR, which also correlates with a longer duration of IFN treatment (> 12 months). The EURO-SKI trial, the largest TKI discontinuation study (758 patients), also showed that patients who receive IFNα prior to TKI maintain TFR. Although now rarely used, physicians may consider IFNα for patients identified at high risk of molecular relapse. The immunomodulatory drug lenalidomide is routinely used to treat multiple myeloma and myelodysplastic syndrome, and stimulates immune effector cells (T cells and NK cells) and blocks MDSCs and Tregs, leading to anti-tumor effects. A small study has shown that lenalidomide potentially contributes to achievement of TFR in patients with CML-CP. Further investigation is warranted to clarify these findings .
Immunomodulatory effects of TKIs
TKIs have well-known off-target immunomodulatory effects. Dasatinib strongly induces expansion of large granular lymphocyte subsets, which have strong cytotoxic functions, yielding favorable clinical outcomes in patients with CML-CP . The proportion of NK-cell subsets with cytotoxic potential increases during imatinib treatment, while no significant immunological changes were observed with bosutinib . Furthermore, treatment with imatinib, dasatinib, or nilotinib reduces immunosuppressive cell fractions, including Tregs and MDSCs . These results suggest that TKIs have immunomodulatory functions, but the association between these off-target effects and TFR has not been fully elucidated.
The immune response in CML have not been fully elucidated comprehensively. We previously reported HLA polymorphisms associated with TFR, while NK cell activation status and KIR polymorphisms contributed to deep molecular DMR [15, 25], thus we hypothesized DMR depends on NK cell activation status and T-cell–mediated immunity contributes to TFR in CML-CP patients.
Several clinical studies show that TKI discontinuation is a safe procedure. The establishment of the optimal treatment strategy before TKI discontinuation is needed for patients who are defined as being at higher risk of molecular relapse.
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This work was supported by research grants from JSPS KAKENHI (19K17860, HU) and Okinaka Memorial Institute for Medical Research (HU).
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Ureshino, H. Treatment-free remission and immunity in chronic myeloid leukemia. Int J Hematol 113, 642–647 (2021). https://doi.org/10.1007/s12185-021-03117-7
- Chronic myeloid leukemia
- Treatment-free remission
- Natural killer cells
- T lymphocytes
- Immune surveillance